This disclosed technology relates to stimulated emission fluorescence and stimulated Raman microscopy and more particularly to in-vivo and in tissue culture stimulated emission.
There is interest in providing deep imaging for use in research, neuroscience, endoscopy, dermatology and intra-surgical definition of clear margins during removal of malignant tissues. For example, Optical Coherence Tomography (OCT) can obtain images up to 1 mm depth in tissue.
In addition there is interest in imaging low concentrations of molecules that do not fluorescence or have week fluorescence such as small drug molecules, metabolic cofactors, and messengers such as ATP and neurotransmitters.
Multi-Photon Excitation (MPE) imaging can enhance the depth of penetration by using infrared photons for excitation where tissue absorption is low. MPE uses two or more photons to excite emission as shown in FIG. 1b. In MPE, the two or more photons can be simultaneously absorbed by one molecule, through the population of one, or more, very short lived virtual states.
MPE excitation has also been used in Fluorescence Lifetime Microscopy (FLIM), for example, to measure the fluorescent lifetimes of bound and free state metabolic cofactor NADH. Fluorescent lifetimes are of importance when determining a metabolic state of cells, in accessing tissue health and differentiating normal from malignant cells. MPE, however, is relatively slow because the fluorescent yield of free NADH is low, has a short excited state lifetime and needs photon counting to create a decay curve.
Standard fluorescence is an incoherent spontaneous emission process where emission of one or multiple photons causes fluorescent emission. In standard fluorescence, the incoherent spontaneous emission can be red shifted from the excitation and can be considered a dark field imaging technique. The measurement process for standard fluorescence is limited to background fluorescence and electrical noise.
Stimulated fluorescent emission (STEM) imaging is a combination of incoherent and coherent processes. (the energy level diagram is shown in FIG. 1a) that uses two photons—a pump and a probe. The pump excites an electron into excited state S1 from ground state S0. A several hundred femtosecond delay, or more, is allowed for the decay of an excited state vibrational level into the lowest excitation level in the excited state manifold S2 via a Kasha decay process. Then a probe (or stimulated emission) beam causes the stimulated emission of a photon and the de-excitation of the electron to S3, which then rapidly decays via a Kasha decay process back to S0. The signal measured is a gain in the probe beam or loss in the pump beam. STEM techniques have been used to image molecules that absorb strongly, but do not fluoresce efficiently such as oxy-hemoglobin, deoxy-hemoglobin, melanin, cytochromes and certain drugs.
STEM is a bright field technique where a signal is added to the forward propagating probe beam. The gain in the beam is 10−4-10−7 (depending on concentration). Therefore, sophisticated electronic signal processing lock-in techniques are usually required to detect a small probe beam change. STEM imaging also uses moderate to high concentrations of molecules to image tissue at moderate to high speed. Unlike fluorescence imaging where emission occurs in any direction, the emission in STEM occurs in the forward direction. Therefore multiple scattering events are required to collect the signal at the tissue surface. STEM is best used for weakly absorbing and scattering tissues but the depth of imaging is limited and requires collection at an angle outside of the imaging aperture, eliminating the ability to do confocal imaging and degrading signal to noise ratio by collecting photons that scatter prior to reaching focus.
Multi-Photon Stimulated Emission Microscopy (MP-STEM) can be used to enhance the depth of penetration and reduce the scattering and absorption of stimulated emission photons in STEM microscopy. MP-STEM uses multiple photons for both excitation and to stimulate emission from weakly fluorescent molecules. The process of MP-STEM can reduce the focal spot size of the emitting region. When using 3 photons or more, the focal spot is reduced in size enough to cause the stimulated emission spot to be small enough to cause dipole-like backscatter emission. This occurs when the axial dimension of the emitting region shrinks to less than 50% of the Stimulated emission wavelength. Dipole backscatter enables enhancement of the detected Signal to Noise Ratio (SNR) because in the backscattered direction the noise is due to the Refractive Index (RI) gradient and MIE scattering from the emission region focus in confocal microscope geometry. This is less than the forward scattered noise normally detected in STEM microscopy, or multiple backscatter STEM detection.
Raman stimulated emission microscope (Ra-STEM) works the same way as single photon fluorescence STEM. Here the excitation occurs in vibrational rather than electronic levels. The mechanism of Ra-STEM is similar to fluorescence STEM, except the excited state produced by the pump is a virtual level of very short lifetime, thus the pump and probe should arrive simultaneously at the sample. Again unless the focal spot is less than ½ the probe wavelength almost all of the stimulated light is scattered in the forward direction.
Thus there are deficiencies in the use of MP-STEM and Raman-STEM                1. The focal spot shrinkage in 1, 2 and 3 photon MP-STEM and 1 photon Raman-STEM is not small enough to provide optimal dipole backscatter.        2. Overlap of converging pump and probe beams may result in stimulated emission outside of the focal region from focal spot enlargement due to refractive index gradients, or relatively high power near focus.        3. Optimal focal spot reduction uses high Numerical Aperture (NA) imaging. This, typically, does not enable large standoff distances that can be desirable for in-vivo imaging or imaging in culture medium.        